Expression of M protein, the major virulence factor of Streptococcus pyogenes is regulated at the transcriptional level by elements in the chromosomal region immediately upstream of the M protein structural gene. One element, designated mry for M protein RNA Yield, has been identified by Tn916 mutagenesis and is located 1.8 kb upstream of both the M protein structural gene and its promoter. However, because of a lack of techniques for genetic analysis in S. pyogenes and gram positive organisms in general, the function of the mry locus and the role of other upstream sequences in regulation is not known. Further study will require the development of new techniques. In this regard, work has begun to develop Tn916-based cloning vectors that generate chimeric "shuttle" transposons for the analysis of cloned genes in S. pyogenes as well as other gram positive organisms. The main features of these vectors are 1: a gene or DNA segment is cloned and manipulated on a plasmid in E.coli; 2: the plasmid construct is transformed into a naturally competent host (such as Bacillus subtilis) to generate the chimeric shuttle transposon; and 3: the conjugative properties of Tn916 are used to mobilize the chimeric transposon into S. pyogenes (or any other gram positive organism). It is even possible to use this system for the allelic replacement of a targeted gene. It is goal of this proposal to develop versions of these vectors that will allow the construction and manipulation of transcriptional fusions to the reporter gene choramphenicol acetyltransferase. These vectors will then be used in a rigorous molecular analysis of the function of the mry locus and other upstream regulatory elements important for expression of M protein and virulence in S. pyogenes. This study will yield important information on gene regulation and regulation of virulence in S. pyogenes and will produce methods that can be applied to the molecular analysis of virulence in any gram positive organism.